1. Field of the Invention
Kamal bunt of wheat is caused by the pathogen Tilletia indica Mitra. The ryegrass smut pathogen, closely related to Tilletia indica, has been isolated from ryegrass seed and recently identified as Tilletia walkeri. This invention relates to novel PCR primers which can be used to detect pathogenic T. indica and T. walkeri and to particularly distinguish T. indica from T. walkeri.
2. Description of the Relevant Art
Tilletia indica Mitra infects wheat, durum wheat, and triticale and causes a disease commonly referred to as Kamal bunt. The disease was first described in the Indian village of Karnal in 1930 (Mitra, M. 1931. Ann. Appl. Biol. 18: 178-179), and since then Karnal bunt has been found in the neighboring countries of Pakistan, Nepal, Iraq, and Afghanistan (Bonde et al. 1996. Plant Dis. 80: 1071-1074). In 1972, Karnal bunt was first reported on the North American continent when it was found in Mexico (Duran, R. 1972. Can. J. Bot. 50: 2569-2573), and by 1982 the disease had become well established in wheat fields in northwestern Mexico (Bonde et al 1997. Plant Dis. 81:1370-1377). Subsequently, in March of 1996, Karnal bunt was discovered in wheat in the United States in Arizona and later that year in California (Bonde et al, 1997, supra; Altschul et al. 1997. Nuc. Acids Res. 25: 3389-3402). In 1997, the disease was discovered in a small area of central Texas (Bonde et al., 1997, supra).
Yield reductions in wheat due to Kamal bunt are slight; however, the disease is of extreme economic importance. Many countries, including the United States, have zero tolerance for Karnal bunt and refuse wheat shipments from quarantined countries and/or areas (Palm, M. E. 1999. Mycologia 91: 1-12). These measures are based on its seed- and soil-borne nature and the lack of effective chemical control methods. Since the U.S. is the world's leading exporter of wheat with an estimated annual value of $5 billion, Karnal bunt poses a serious threat to international trade for the U.S. wheat industry (Castlebury et al. 1999. Mycologia 91: 121-131; Palm, supra). It is therefore desirable to control Kamal bunt at two levels (1) to manage the disease where it occurs so that losses in yield and quality are minimized, and (2) to contain the disease or contaminating teliospores for trade or regulatory purposes (Bonde et al., supra).
Prior to the discovery of Kamal bunt in the U.S., PCR primers were developed to distinguish T. indica from other smut fungi, such as T. barclayana (sometimes referred to as T. horrida) which causes rice kernel smut, that are sometimes present as contaminants in harvested or stored wheat. The technique using PCR primers selected from mitochondrial DNA sequences has been especially useful for differentiating free teliospores of T. indica and T. horrida that are in the same size range. For example, DNA extracted from mycelia obtained from germinated teliospores washed from wheat seeds can be quickly tested using the primers TI17M1/M2 and TI57M1/M2 (Smith et al. 1996. Phytopath. 86: 115-122) and the primers Ti-1/Ti-4 (Ferreira et al. 1996. Appl. Environ. Microbiol. 62: 87-93). The primers TI17M1/M2 and TI57M1M2 tested positive with over 78 isolates of T. indica and negative with 69 isolates of T. horrida. These PCR primers were used to confirm the initial discovery of teliospores in the southwestern U.S. in 1996 as T. indica.
During the 1996 National Kamal Bunt Survey conducted by the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture, teliospores that morphologically resembled T. indica were discovered as free spores in seed washes of wheat from the southeastern United States and in ryegrass seed lots from Oregon (Bonde et al., 1997, supra; Cunfer et al. 1999. Plant Dis. 83: 685-689; Palm, supra). Our existing PCR primers were not able to differentiate between this newly discovered ryegrass smut pathogen and T. indica. Recently, the ryegrass smut was described as a new Tilletia species, T. walkeri, based upon differences in teliospore morphology and ornamentation (Castlebury et al., supra). Since the morphological differences that distinguish teliospores of T. indica from T. walkeri require examination of several spores by trained mycologists, this process can be tedious and subject to misinterpretation. Concerns exist that U.S. wheat shipments could be rejected at international ports due to the misidentification of T. walkeri teliospores as T. indica.
In view of the importance of accurate identification of T. indica because of the potential risk of its dissemination in international exchange of germplasm and in international trade, there is a need for improved primers and diagnostic tests which distinguish between T. indica and T. walkeri, isolated from ryegrass seed, to facilitate implementing specific disease control strategies and for accurately selecting areas for quarantine. Such methods and reagents are valuable tools for monitoring natural disease spread, tracking the soil- and seed-borne fungus in field studies, and detecting the presence of the fungus in grains entering T. indica-free areas.
5'-fluorogenic TaqMan PCR assays have been used to detect other pathogenic microorganisms, including plant pathogenic bacteria (Schaad et al. 1999. Plant Dis. 83: in press), and viruses (Schoen et al. 1996. Phytopath. 86: 993-999), and human fungal pathogens (Brandt et al. 1998. J. Clin. Microbiol. 36: 2057-2062; Shin et al 1999. J. Clin. Microbiol. 37: 165170); however, this is the first application of the TaqMan system for the detection and identification of plant pathogenic fungi. Since serious economic consequences can result from either erroneously identifying a pathogenic organism or subjecting large agricultural areas to quarantine unnecessarily, the primers, reagents, and assays of the invention will be useful in alleviating discrepancies or resolving disputes that might arise regarding the identity of smut teliospores in wheat shipments.